The islets of Langerhans are responsible for maintaining blood glucose levels through insulin secretion. In -cells, insulin release is initiatd by metabolic and electrical events, in particular an influx of calcium into the cell following membrane depolarization. Connexin36 (Cx36) gap junctions provide electrical coupling between -cells, which coordinates intracellular calcium signaling across the islet to coordinate insulin release. Knockout of islet Cx36 gap junctions disrupts this coordination and elevates [Ca2+]i, leading to reduced first phase insulin secretion and diminished second phase pulses, similar to that seen in pre-diabetes. Pro- inflammatory cytokines have been implicated as mediators of -cell dysfunction in early diabetes and in islet transplant surgery. Preliminary data has shown that exposure of -cells to low levels of pro-inflammatory cytokines, similar to those seen in early diabetes alters calcium signaling and impairs insulin secretion dynamics. Taken together, this suggests a role for gap junction coupling in contributing to islet dysfunction during the pathogenesis of diabetes. We hypothesize that pro-inflammatory cytokines decrease gap junction coupling in islets via nitric oxide mediated channel closure, leading to islet dysfunction and increased calcium mediated mitochondrial stress, while GLP-1R mediated increases in cAMP will increase gap junction coupling through Epac2 and PKA regulatory pathways. To test this, we propose to do a series of in vitro studies, where isolated mouse and human islets, islets with a genetic knockout of Cx36, or islets treated with Cx36 inhibitors/activators, will be culture with varying levels of pro-inflammatory cytokines. Islet function will be assessed through calcium signaling, insulin dynamics, Cx36 gap junction coupling and mitochondrial function, including the use of advanced microscopy approaches. The role of nitric oxide and cAMP on gap junction coupling will be determined through culture with chemical inhibitors/agonists of the proposed mediators and analysis of Cx36 phosphorylation, trafficking and membrane plaque formation. The effects of pro- inflammatory cytokines on gap junction coupling in the islet could represent a previously unknown mechanism of islet dysfunction contributing to the pathogenesis of diabetes. Understanding the mechanisms involved in regulating gap junctions could help to identify targets for potential therapies in Type 1 and Type 2 Diabetes as well as ways to improve graft survival and function in islet transplant surgeries.